Azahydroxybenzotriazoles and derivatives thereof for peptide coupling reactions

ABSTRACT

This invention relates to a process for forming an amide or an ester from a reaction between an amine or an alcohol, respectively and an acylating derivative of a carboxylic acid, in the presence of an effective amount of a compound having the formula:   &lt;IMAGE&gt;   and N-oxides thereof and salts thereof.

GOVERNMENT SUPPORT

This work has been supported by a grant from the National Institutes ofHealth GM-09706 and the National Science Foundation (CHE-9003192). TheGovernment has certain rights in the invention.

RELATED APPLICATION

This is a continuation-in-part of copending U.S. patent application Ser.No. 952,025, filed on Sep. 28, 1992 now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a new process for effecting theacylation step in amide formation, especially during peptide synthesis.More specifically, the invention relates to the use of a compound havingthe formula ##STR2## and N-oxides thereof and salts thereof wherein

R₁ and R₂ taken together with the carbon atoms to which they areattached form a heteroaryl ring wherein said heteroaryl ring is anoxygen, sulfur or nitrogen containing heteroaromatic containing from 3and up to a total of 13 ring carbon atoms, said heteroaryl may beunsubstituted or substituted with lower alkyl or an electron-donatinggroup;

Y is O, NR₄, CR₄ R₅ ;

R₄ and R₅ are independently hydrogen or lower alkyl;

X is CR₆ R₇ or NR₆ ;

R₆ and R₇ are independently hydrogen or lower alkyl; or R₆ and R₇ takentogether form an oxo group or when n=O, R₄ and R₆ taken together mayform a bond between the nitrogen or carbon atom of Y and the nitrogen orcarbon atom of X;

Q is (CR₈ R₉) or (NR₈);

when n is 1, R₄ and R₈ taken together may form a bond between the ringcarbon or nitrogen atom of Q and the ring carbon or nitrogen atom of R₈;

n is 0, 1 or 2;

R₃ is hydrogen, lower alkyl carbonyl, aryl carbonyl, lower aryl alkylcarbonyl, AA₁ -BLK₁, a positively charged electron withdrawing group,SO₂ R₁₄, or ##STR3##

R₁₄ is lower alkyl, aryl or lower arylalkyl; q is 0-3;

R₈ and R₉ are independently hydrogen or lower alkyl or R₇ and R₈ takentogether with the carbon to which they are attached form an aryl ring,AA₁ is an amino acid and BLK is an amino protecting group, and m is 0 or1.

The present invention also relates to novel compounds encompassed by theabove-identified formula.

2. Description of the Prior Art

Polypeptides are useful as medicaments. In recent years, peptides havebeen found to be useful in combatting various diseases, such as cancer,diabetes, plant toxins and the like. Additionally, peptides have shownspecific activity as growth promoters, suppressants, antibiotics,insecticides, contraceptives, anti-hypertensives, sleep-inducers,anti-depressants, analgesics, etc. The list is long and varied.

As more and more polypeptides become of medicinal importance, there isan increasing incentive to improve the methods by which they may besynthesized. Currently, syntheses of peptides are in solution byclassical or various repetitive methods. Alternatively, peptides may beprepared on a solid support (Merrifield method). These are all populartechniques in synthesizing peptides from the coupling of two or moreamino acids, in synthesizing larger peptides from the coupling of aminoacids with smaller peptides or in the coupling of smaller peptides.Solution methods have the advantage of being easily monitored, allowingpurification of intermediates, if necessary, at any stage. A majordrawback, however, is the relative slow pace of synthesis, with eachstep being carried out manually.

The major advantage of the Merrifield method is its easy automation sothat unattended, computer-controlled machine synthesis is possible.Unfortunately, the method suffers from an inherent deficiency due to theinsoluble nature of the support on which the synthesis proceeds. Unlesseach acylation step occurs with approximately 100% efficiency, mixtureswill inevitably be built up on the polymer. The longer the chain, thegreater will be the contamination by undesired side reactions. Sideproducts produced in such reactions remain to contaminate the desiredproduct when it is removed from the polymeric matrix at the end of thecycle. These current techniques are not useful in preparing peptides ofgreater than 20-30 residues; separation of side products from thedesired product becomes increasingly difficult when larger peptides aresynthesized.

For very long segments (50 or more amino acids), therefore, currentmethods are not satisfactory. Often, mixtures are obtained of suchforbidding complexity that it may be difficult or impossible to isolatethe desired peptide.

The problems enumerated hereinabove may be eliminated if the properderivatives of the underlying amino acids and/or the proper conditionsfor the coupling reaction could be found. Protecting groups, such ast-butyloxy-carbonyl (t-Boc) or N-α-(9-fluorenylmethyl)oxycarbonyl(Fmoc), have been used to minimize side reactions. But, additionally,other aspects of the coupling reaction must also be taken intoconsideration, such as the peptide coupling additive to be used in thecoupling reaction.

Additives generally inhibit side reactions and reduce racemization.Heretofore, the most common peptide coupling additive used duringpeptide coupling for both solutions and solid phase syntheses is1-hydroxybenzotriazole (HOBt). This reagent has been used either incombination with a carbodimide or other coupling agent or built into astand-alone reagent, such as1-benzotriazolyoxytris(dimethylamino)phosphonium hexafluorophosphate(BOP) or an analogous uronium salt. HOBt is applicable to both stepwiseand segment condensations. However, many cases have been encountered inwhich HOBt is ineffective, possibly because of steric effects, or lowbasicity of the amino component. Especially problematic are segmentcouplings at amino acid units other than glycine or proline, since theproblem of racemization may be severe. The relatedN-hydroxybenzotriazinone (HOOBt) may provide better protection againstracemization, but it is rarely used due to competing side reactionsinvolving ring openings.

However, the present inventor has discovered that compounds of Formula Iare effective as peptide coupling additives in both stepwise (batch andcontinuous flow) and segment condensations to peptide syntheses.Compounds of Formula I overcame deficiencies of the additives usedheretofore. Compounds of the present invention, as a peptide couplingadditive, have the ability to accelerate the reaction, or providecleaner processes, higher yields and less racemization. The productsformed with the use of compounds of the present invention tend to bepurer than those made by methods used heretofore. Yet, the reactionconditions are very mild, and the reagents used are commerciallyavailable and/or easy to prepare.

Furthermore, compounds within the scope of the present invention have anadditional benefit and provide a visual indication of the reactionendpoint. For example, HOAt or 1-hydroxy-7-azabenzotriazole in thepresence of an amino acid or peptide ester, is converted to its anion,which is colored. As coupling proceeds, the color fades and thendisappears completely when the coupling reaction is completed. Bywatching for the disappearance of the color, the researcher knows whenthe coupling reaction is completed. The researcher does not need tomonitor the reaction to determine when the coupling reaction iscompleted. He does not need to wait an unspecified amount of time forthe reaction to be completed. Thus, by using the compound of the presentinvention the researcher, as a result, can use his time moreefficiently.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to the use of a compound of Formula I inpeptide synthesis in the preparation of a peptide bond. Morespecifically, the present invention relates to a process for preparing apeptide bond which comprises reacting a first amino acid or a firstpeptide each having a free amino group with a second amino acid or asecond peptide, each having a free carboxy group or an acylatingderivative thereof in the presence of an effective amount of a compoundhaving Formula I under amide forming conditions. In addition, thepresent invention is directed to novel compounds of Formula I.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As described hereinabove, an embodiment of the present invention relatesto compounds of Formula I and their use in peptide coupling. In otherwords a first amino acid or a first peptide, each having a free aminogroup is coupled with an acylating derivative of either a second aminoacid or a second peptide in the presence of compounds of Formula I underamide forming conditions to form a peptide bond and thus form a largerpeptide.

As employed herein, the term "heteroaryl" is a heteroaromatic containingat least one heteroatom ring atom selected from nitrogen, sulfur andoxygen and up to a maximum of four ring heteroatoms. The heteroarylcontains from 5 to 14 ring atoms and up to a total of 13 ring carbonatoms and a total of 18 carbon atoms. The heteroaryl group may bemonocyclic, bicyclic or tricyclic. Also included in this expression arethe benzoheterocyclic. The heteroaryl group preferably contains no morethan two ring heteroatoms, and most preferably contains one ringheteroatom. The most preferred ring heteroatoms are oxygen and nitrogen,with nitrogen being the most preferred.

If nitrogen is a ring atom, N-oxides can also be formed. The presentinvention contemplates the N-oxides of the nitrogen containingheteroaryls.

Examples of heteroaryls include thienyl, benzothienyl, 1-naphthothienyl,thianthrenyl, furyl, benzofuryl, pyrrolyl, imidazolyl, pyrazolyl,pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, isoindolyl,indazolyl, purinyl, isoquinolyl, quinolyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, carbolinyl, isothiazolyl,isoxazolyl and the like. It is preferred that the heteroaryl group ispyridyl, pyrrolyl, furyl, indolyl, quninolyl, isoquinolyl or benzofuryl.Especially preferred is pyridyl.

When R₁ and R₂ taken together with the carbons to which they areattached form a tricyclic heteroaryl group, then the compounds ofFormula I is tetracyclic; if a bicyclic heteroaryl group is formed fromR₁ and R₂ taken together with the carbons to which they are attached,then the compounds of Formula I are tricyclic. Finally, if R₁ and R₂taken together form a monocyclic heteroaryl group, then the compounds ofFormula I are bicyclic. It is preferred that compounds of Formula I aretricyclic, and especially bicyclic.

The term "alkyl", when used alone or in combination with other groups,refers to a carbon chain containing from one to six carbon atoms. It maybe a straight chain or branched and includes such groups as methyl,ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, t-butyl,n-pentyl, amyl, hexyl and the like. The preferred alkyl group containsfrom 1-3 carbon atoms, and most preferably methyl.

The term "aryl" as used herein, alone or in combination, refers to anaromatic ring system containing from 6-10 ring carbon atoms and up to atotal of 15 carbon atoms. It includes such groups as phenyl, α-naphthyl,β-naphthyl and the like.

Aralkyl groups are aryl groups attached to the main chain through analkylene bridge. Such groups include benzyl, phenethyl and the like.

Alkyl carbonyl refers to an alkyl group attached to the main chainthrough a carbonyl. Similarly, aryl carbonyl refers to an aryl groupattached to the main chain through a carbonyl group.

As used herein, an "electron donating group" shall designate a groupthat will release or donate electrons more than hydrogen would if itoccupied the same position in the molecule. See J. March, AdvancedOrganic Chemistry, 3rd Ed., John Wiley & Sons p. 238 (1985). These typesof groups are well known in the art. Examples include lower alkylamino,diloweralkylamino, amino, halo, aryl, lower alkoxy, lower aralkoxy,aryloxy, mercapto, lower alkylthio, and the like. The preferred electrondonating groups are amino, hydroxy, lower alkoxy, lower alkylamino anddiloweralkylamino.

The term "electron withdrawing groups" as defined herein refer to agroup that will draw electrons to itself more than a hydrogen atom wouldif it occupied the same position in the molecule. See, J. March,Advanced Organic Chemistry, 3rd Ed., John Wiley & Sons P. 17 (1985).They include such groups as nitro, monohaloalkyl, dihaloalkyl,trihaloalkyl (e.g., CF₃), halo, formyl, lower alkanoyl, loweralkylsulfonyl, lower alkylsulfinyl, and the like.

A positively charged electron withdrawing group is an electronwithdrawing group bearing a positive charge and forming a stable bond toa N-hydroxide (N-0). These types of groups are well known in the art.Examples include uronium groups, e.g., ##STR4## imino cations e.g.,##STR5## phosphonium cations, e.g., --P(NR₁₂ R₁₃)₃ and the like, whereinR₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen or lower alkyl, loweralkoxy lower alkyl or R₁₀ and R₁₂ taken together with the atoms to whichthey are attached form a ring containing up to 6 ring atoms and up to atotal of 5 ring carbon atoms or R₁₂ and R₁₃ taken together with thenitrogen atom to which they are attached may form a 5 or 6 memberedheterocyclic ring containing up to a total of 5 ring carbon atoms. It ispreferred that R₁₀ and R₁₁ and R₁₂ and R₁₃, when both are present, arethe same. It is especially preferred that R₁₀, R₁₁, R₁₂, R₁₃, when everare present, are the same.

Preferred cyclic uronium and imino groups have the formula ##STR6##respectively, wherein R₁₁ and R₁₂ are as defined hereinabove and n is 0or 1.

In the above formulae, the preferred values of R₁₀, R₁₁, R₁₂ and R₁₃ aremethyl, ethyl, n-butyl, pentyl and --CH₂ CH₂ --O--CH₂ CH₃. It ispreferred that R₁₁ and R₁₂ are the same.

The preferred values of R₁₂ and R₁₃ are lower alkyl, especially methyl.It is preferred that both R₁₂ and R₁₃ are both the same. Further, it ispreferred that both are methyl.

When R₁₂ and R₁₃ taken together form a ring, they may form heterocyclicmoleties of the formula: ##STR7## wherein U=CH₂, O, or N-Alk, whereinAlk is lower alkyl, especially methyl.

It is preferred that R₇ and R₈ are hydrogen or lower alkyl, but mostpreferably hydrogen.

Preferred values of Y are S, O, NR₄ or CR₄ R₅, wherein R₄ and R₅ areindependently hydrogen or methyl. Especially preferred values of Y are0, CH₂ or NH.

It is preferred that X is CR₆ R₇ or NR₆. Preferred values of R₆ and R₇are hydrogen or lower alkyl. When R₆ and R₇ taken together form an oxogroup, X becomes C=O. It is most preferred that X is C=O, CH₂ or NH orN(CH₃). However, in cases when n is 0, then R₄ and R₆ taken together mayform a bond between X and Y, i.e., a bond may form between the ringcarbon atoms of X and the ring carbon atom of Y, or between the ringnitrogen atom of X and the ring nitrogen atom of X, or the ring nitrogenatom of X, and the ring carbon atom of Y or the ring carbon atom of Xand the ring nitrogen atom of Y. In other words, under thesecircumstances, the compound of Formula I becomes ##STR8## wherein R₁,R₂, Y, X, and R₃ are as defined above. Under these circumstances, it ispreferred that Y is CH or N and X is CH or N. It is most preferred thatY and X are N.

When n is 1, the compound of Formula I becomes ##STR9## wherein

Q, R₁, R₂, Y, R₈, R₉ X and R₃ are as defined above. It is preferred thatR₈ e and R9 are hydrogen.

As indicated hereinabove, when n is 1, R₄ and R₈ taken together may forma bond between Q and Y, i.e., the ring carbon atom of R₄ and the ringcarbon atom of R₈ may form a bond, or the ring carbon atom of R₄ and thering nitrogen atom of R₈ may form a bond, or the ring nitrogen atom ofR₄ and the ring carbon atom of R₈ may form a bond or the ring nitrogenatom of R₄ and the ring nitrogen atom of R₈ may form a bond. In otherwords, under these circumstances, the formula becomes: ##STR10##

Preferred values of Y in this formulation are S, O CH₂ or N--H orN--(CH₃). It is most preferred under these circumstances that Y is S, Oor N(CH₃).

The preferred values of X in this formulation are C═O or NH or CH₂.

Preferred values of Q are CH₂ or NH. However, it is also preferred thatthe above-identified compound has the formula: ##STR11## wherein

Q is CR9 or N,

R₉ is hydrogen or lower alkyl and R₁, R₂, X, OR₃ are as definedhereinabove. Examples of the above formula include: ##STR12## etc.

It is also preferred that compounds of Formula I have the formula:##STR13## wherein R₁, R₂, R₃, Y and X are as defined hereinabove.

Preferred compounds of Formula I have the formula: ##STR14## or N-oxidesthereof wherein Q, Y, X, R₃, n, R₄, R₅, R₆, R₇, R₈, R₉ and R₁₄ are asdefined hereinabove,

A is N or CR₁₅ ;

D is CR₁₆ or N;

E is CR₁₇ or N;

G is CR₁₈ or N; and

R₁₅, R₁₆, R₁₇ and R₁₈ are independently hydrogen or lower alkyl or anelectron donating group or R₁₆ and R₁₇ taken together form an aryl ring,but at least one of A, D, E, G is N.

It is preferred that no more than two of A, D, E, G are N. It is mostpreferred that only one of A, D, E, G is N. Further, it is preferredthat R₁₅, R₁₆, R₁₇ or R₁₈ are hydrogen or an electron-donating group, asdefined herein. The preferred electron donating group is lowerdialkylamino especially N, N-dimethyl-amino and lower alkoxy, e.g.methoxy.

Preferred compounds of Formula II have the formulae: ##STR15## orN-oxides thereof wherein Y, X, n, Q and R₃ are as defined hereinaboveand R₁₅ and R₁₇ are independently lower alkyl and more preferablyhydrogen or an electron donating group.

Of the compounds of Formula III-VI, the most preferred compound is thatof Formula IIIa ##STR16## or N-oxides thereof wherein Q, Y, x and R3 areas defined hereinabove and R₁₅ and R₁₇ are lower alkyl or hydrogen or anelectron withdrawing group.

Preferred compounds of Formula I also have the formula ##STR17## orN-oxides thereof wherein R₈, R₉, n, Q, D, E, X and Y are as definedhereinabove and J is NR₁₅, O, CR₁₅ R₁₉ or S(O)p, and p is 0, 1, 2.

R₁₅ is as defined hereinabove and R₁₉ is hydrogen or lower alkyl. It ispreferred that R₁₉ is hydrogen, and preferred values of R₁₅ is anelectron donating group or hydrogen.

Preferred values of J are O or S(O)p; the preferred value of p is 1.

Preferred compounds of Formula VII have the formula: ##STR18## orN-oxides thereof wherein J, Y, R₈, R₉ n and R₃ are as definedhereinabove and X is C=O.

In compounds VII, VIII or VIIa as depicted above, it is preferred thatat least one of D, E or J is a heteroatom. Furthermore, it is mostpreferred that at most two of J, E and D are heteroatoms. It is mostpreferred that only one of J, E and D is a heteroatom.

Thus, the present invention includes compounds having the formula:##STR19## or N-oxides thereof wherein A, D, E, G, Y, X, R₃ and J are asdefined hereinabove.

Furthermore, the present invention includes use of compounds of theformula ##STR20## or N-oxides thereof.

In the above formulae, when the ring contains Y=X, this means that R₄ ofY and R₆ of X are joined together to form a ring bond between the Y ringatom and the X ring atom, so that as depicted hereinabove there is adouble bond between the Y ring atom and the X ring atom. Furthermore, inthe above formulae, when the ring contains Y═N, then R₄ of Y and R₈ ofNR₈ of Q joined together to form a ring bond so that there is a doublebond between the nitrogen ring atom and the Y atom. Thus, Y is CR₅ or Nunder these circumstances.

Preferred embodiments of compounds of Formula I include ##STR21## or theN-oxides thereof wherein ##STR22## R₁₀ and R₁₂ are independently methyl,ethyl, propyl, butyl, pentyl CH₂ CH₂ O--CH₂ CH₃,

R₁₅ is Me, Et, i-Pr, iPr₂ N, or CMe₃

J is O, or S(O)p, and p is 0, 1 or 2.

Of course, various combinations and permutations of the formulaedescribed herein are also contemplated by the present invention. Inaddition, Markush groupings containing less than all of the elementsdescribed hereinabove as well as the various permutations thereof arealso contemplated by the present invention.

As described herein, the compounds described hereinabove are useful inpromoting peptide coupling, i.e., the reaction between a free aminogroup of a first amino acid or first peptide with a free carboxy groupor acylating group of a second amino acid or peptide. The process of thepresent invention is general; it can be used in effecting the couplingof a dipeptide and an amino acid, a tripeptide and an amino acid, atetrapeptide and an amino acid, dipeptides, pentapeptides, higherpeptides, polypeptides etc.

When the compound of Formula I reacts with an amino compound such as anamino blocked amino acid or protein of the formula BLK₁ -AA₁ thecorresponding amino acid ester of the formula is formed, i.e., ##STR23##wherein AA₁ is an amino acid or protein as defined herein, BLK₁ is ablocking group as defined herein and Y, Q, n, x, R₁, and R₂ are asdefined hereinabove. This amino acid ester can then react with acompound having a free amino, such as an arylamino, alkylamino, loweraryl amino, etc. designated as R₂₁ R₂₂, NH, wherein R₂₁ and R₂₂ areindependently hydrogen, lower alkyl, aryl or lower aryl alkyl to form acompound of the formula: ##STR24## Removal of the blocking group bytechniques known to one skilled in the art affords the product:

    AA.sub.1 NR.sub.21 R.sub.22.

This technique is extremely useful when the second amino compound is anamino acid or peptide having a free amine group, designated as AA₂. Inthis case, a peptide is formed between AA₁ and AA₂ ; for example,##STR25## wherein AA₁, AA₂, BLK₁, R₁, R, Y, Q, n and X are as definedherein.

As with most peptide coupling reactions, a dehydrating agent, such asEDC or DCC can be present.

The blocking group can be any of the blocking groups described herein,but the preferred blocking groups are FMOC, BOC, benzyloxycarbonyl BSMOCand Bspoc.

The term "amino acid" or AA, AA₁, or AA₂ as used herein refers to anorganic acid containing both a basic amino group (NH₂) and an acidiccarboxyl group. (COOH). Therefore, said molecule is amphoteric andexists in aqueous solution as dipole ions. (See "The Condensed ChemicalDictionary", 10th Ed., edited by Gessner G. Hawley, Van NostrandReinhold Company, London, England p. 48 (1981). The preferred aminoacids are the α-amino acids. They include but are not limited to the 25amino acids that have been established as protein constituents. Theymust contain at least one carboxyl group and one primary or secondaryamino group in the amino acid molecule. The term includes suchproteinogenic amino acids as alanine, valine, leucine, isoleucine,norleucine, proline, hydroxyproline, phenylalanine, tryptophan, amino,subutryic acid, methionine, glycine, serine, threonine, cysteine,cystine, glutamic acid, lysine, hydroxylysine, ornithine, arginine,histidine, penicillamine, naphthylamine, α-phenylglycine, and the like.

As used herein, the term "peptide" refers to the class of compoundscomposed of amino acid units chemically bound together with amidelinkages. A peptide may contain as little as two amino acid residues ormay contain a polymer of amino acid residues (polypeptide).

As used herein, the terms "amino acid" and "peptide" also include aminoacids and peptides, respectively containing blocking (protecting)groups. These protecting "groups" block the amino group or the carboxylgroup of the amino acid or peptide not involved in or taking part in thecoupling in order to prevent unwanted side reactions. These protectinggroups also protect reactive groups on the side chain.

A number of blocking reagents for amino groups are known in the art andhave been utilized in the syntheses of peptides. These blocking groupsare discussed in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396,4,581,167, 4,394,519, 4,460,501 and 4,108,846, the contents of all ofwhich are incorporated by reference as if fully set forth herein. Otheramino protecting groups are discussed in U.S. patent application Ser.No. 364,662, the contents of which are also incorporated by reference.Other amino protecting groups are described in an article entitled"Solid Phase Peptide Synthesis", by G. Barany and R. B. Merrifield inTHE PEPTIDES, Vol. 2, edited by E. Gross and J. Meienhoffer, AcademicPress, N.Y., N.Y. 100-118 (1980), and in the book entitled "PROTECTIVEGROUPS IN ORGANIC SYNTHESIS" by T. W. Green, John Wiley & Sons, NewYork, the contents of all of which are being incorporated by reference.

The term amino acid protecting group, (BLK, BLK,) as used herein, refersto blocking groups which are known in the art and which have beenutilized to block the amino (NH₂) group of the amino acid. Blockinggroups such as 9-loweralkyl-9-fluorenyloxycarbony,2-chloro-1-indanylmethoxy-carbonyl (CLIMOC)and benz [f] indene-3methyloxycarbonyl (BIMOC) and dbd-TMOC arediscussed in U.S. Pat. Nos. 3,835,175, 4,508,657, 3,839,396, 4,581,167,4,394,519, 4,460,501 and 4,108,846 referred to hereinabove. Moreover,other amino protecting groups such as 2-(t-butylsulfonyl)-2-propenyloxycarbonyl (Bspoc) and benzothiophenesulfone-2-methoxycarbonyl (Bsmoc) are discussed in copendingapplication, U.S. Patent Application Ser. No. 364,662, the contents ofwhich are incorporated herein by reference. Other N-amino protectinggroups include such groups as the t-butyloxycarbonyl (BOC),t-amyloxycarbonyl (Aoc), β-trimethylsilyl-ethyloxycarbonyl (TEOC),adamantyl-oxycarbonyl (Adoc), 1-methylcyclobutyloxycarbonyl (Mcb),2-(p-biphenylyl)propyl-2-oxycarbonyl (Bpoc),2-(p-phenylazophenyl)propyl-2-oxycarbonyl (Azoc),2,2-dimethyl-3,5-dimethyloxybenzyloxycarbonyl (Ddz),2-phenylpropyl-2oxycarbonyl (Poc), benzyloxycarbonyl (Cbz),p-toluenesulfonyl aminocarbonyl (Tac), o-nitrophenylsulfenyl (Nps),dithiasuccinoyl (Dts), Phthaloyl, piperidineoxycarbonyl, formyl,trifluoroacetyl and the like.

These protecting groups can be placed into four categories:

1) a base labile Nα-amino acid protecting group such as FMOC, and thelike.

2) protecting groups removed by acid, such as Boc, TEOC, Aoc, Adoc, Mcb,Bpoc, Azoc, Ddz, Poc, Cbz, 2-furanmethyloxycarbonyl (Foc),p-methoxybenzyloxycarbonyl (Moz), Nps, and the like.

3) protecting groups removed by hydrogenation such as Dts, Cbz.

4) protecting groups removed by nucleophiles, such as Bspoc, Bsmoc andNps and the like.

5) protecting groups derived from carboxylic acids, such as formyl,acetyl, trifluoroacetyl and the like, which are removed by acid, base ornucleophiles.

A variety of carboxy protecting groups known in the art may be employed.Examples of many of these possible groups may be found in "ProtectiveGroups in Organic Synthesis", by T. W. Green, John Wiley & Sons, 1981,the contents of which is incorporated by reference. These examplesinclude such groups as methyl ester, t-butyl ester,β-trimethylsilylethyl ester, benzyl ester and the like.

In addition, during the course of protein synthesis, it may be necessaryto protect certain side chains of the amino acids to prevent unwantedside reactions. The various protecting groups are discussed in copendingU.S. patent application No. 426,121, the contents of which areincorporated herein by reference.

The term "acylating group of an amino acid or peptide" refers to a groupon the free carboxy end of the amino acid or peptide that facilitatesthe acylation reaction, i.e., nucleophilic substitution at the acylcarbon. Examples include the free acid, acid halide, esters, such aslower alkyl esters, phenoxy esters which are unsubstituted orsubstituted with 1-5 electron withdrawing groups as defined herein; oran anhydride and the like. The preferred acylating derivative is theacid, acid halide, especially the acid chloride or fluoride, and thephenoxy ester.

The preferred acylating amino acid is an amino acid group of the formula

    BLK-AA-M,

wherein BLK is an amino protecting group

AA is an amino acid and

M is halo or ##STR26## wherein R₂₀ is independently halo, lower alkyl,nitro, cyano or other electron withdrawing groups and n is 0-5. When nis 0, the phenoxy ester is unsubstituted.

The most preferred acylating group of an amino acid is the amino acidchloride or fluoride. The preparation and use of amino acid chlorides asan acylating derivative is discussed in an article by Carpino, et al. inJ. Org. Chem., 1986, 51, 3734-3736, the contents of which areincorporated herein by reference. Briefly, amino acid chlorides can beprepared by reacting the amino acid with thionyl chloride andrecrystallizing the product from a recrystallization reagent, such asCH₂ Cl₂ -hexane.

The preparation and use of amino acid fluorides in peptide synthesis arediscussed in copending U.S. patent application having Ser. No. 426,121,the contents of which are incorporated herein by reference. As describedtherein, the amino acid fluorides can be prepared by reacting anN-protected amino acid with the reagent cyanuric fluoride. This reactioncan be run at temperatures as low as 0° C. and up to the refluxingtemperature of the solvent, but it is preferred that the reaction is runat room temperature. It can also be run in an inert solvent, such aspyridine/CH₂ Cl₂ and the like. The cyanuric fluoride can be preparedfrom the corresponding chloride in the presence of potassium fluoride atelevated temperatures ranging from 150° to 250° C., according to thefollowing equation ##STR27##

Other fluorinating agents well known in the art, such as thionylfluoride, 2,4,6-trinitrofluorobenzene, N-methyl-2-fluoropyridiniumsalts, and the like may be used in place of KF to effect the formationof cyanuric fluoride.

A typical preparation of the peptide in accordance with the presentinvention involves the following steps

1) protection of the free carboxyl group in a first amino acid or afirst peptide, unless the amino acid or peptide is anchored to a solidsupport.

2) protection of the free amino group of a second amino acid or peptide.

3) protection of the side chains, if necessary.

4) coupling the first amino acid or peptide with the second amino acidor peptide in the presence of compounds of Formula I.

5) removal of the protecting groups.

The procedure of steps 1-3 can be performed in any order.

In the coupling step, the compounds of Formula I should be present ineffective amounts. Usually, the first amino acid or peptide is presentin approximately equimolar amounts with the second amino acid orpeptide, although the reaction can take place if the molar ratio of theformer to the latter ranges from 1:3 to 3:1. Furthermore, the amount ofthe compound having Formula I used depends upon the amount of peptide oramino acid which is present in the least amount (i.e. the limitingreagent); thus the molar ratio of the compound of Formula I to the aminoacid or peptide ranges from 1:3 to 3:1 relative to the amino acid orpeptide present in the least molar amount, although it is preferred thatapproximately equimolar amounts of the compound of Formula I, the firstamino acid or peptide and the second amino acid or peptide be used.

The coupling reaction described hereinabove can take place in theadditional presence of a dehydrating agent such as DCC(dicyclohexylcarbodiimide) or EDC,(N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride) and thelike. The coupling reaction usually takes place in an inert organicsolvent such as dimethylformamide (DMF) or ethers, such as ethyl ether,THF or dioxane. In fact DMF is the preferred solvent in the solid phasesynthesis because of its favorable solvation properties. The reactiontakes place under mild conditions usually ranging from about 0° C. toabout 30° C. After the peptide is formed, the blocking groups areremoved by techniques known to one skilled in the art.

The following sequence is illustrative of the coupling reaction; in theexamples below, amino acids (AA) are used, although the procedure isgeneral for amino acids and/or peptides: ##STR28##

In the above scheme, BLK is an amino acid blocking group, AA₁, AA₂ andAA₃ are first, second and third amino acid, respectively and P is acarboxy protecting group.

As shown by the above scheme, the N-αamino protected amino acid isreacted with a second amino acid in which the carboxy group isprotected.

A peptide is formed between the first amino acid and the second aminoacid. The peptide chain can be increased by removing the alpha aminoprotecting group by techniques known to one skilled in the art and thenreacting the corresponding dipeptide with another N-α amino protectedamino acid in the presence of a compound of Formula I to form thecorresponding tri-peptide. The N-α amino protecting group of thetri-peptide is removed and the above-cycle is repeated until the desiredpeptide has been obtained.

The present invention can readily be utilized in solid phase peptidesynthesis. Solid phase peptide synthesis is based on the stepwiseassembly of a peptide chain while it is attached at one end to a solidsupport or solid phase peptide resin. Two methods are generally wellknown in the art.

One, the Merrifield method, employs a solid support for attachment ofthe amino acid or peptide residues. This method employs N-protectedamino acids as building blocks which are added to an amino acid orpeptide residue attached to the solid support at the acyl (acid) end ofthe molecule. After the peptide bond has been formed, the protectedgroup is removed and the cycle repeated. When a peptide having thedesired sequence has been synthesized, it is then removed from thesupport.

The second method, the inverse Merrifield method, employs reagentsattached to solid supports in a series of columns. The amino acid orpeptide residue is passed through these columns in a series to form thedesired amino acid sequence.

These methods are well known in the art as discussed in U.S. Patent Nos.4,108,846, 3,839,396, 3,835,175, 4,508,657, 4,623,484, 4,575,541,4,581,167, 4,394,519 as well as in Advances in Enzymology, 32, 221(1969) and in PEPTIDES, Vol. 2, edited by Erhard Gross and JohannesMeienhoffer, Academic Press, New York pp. 3-255 (1980) and the contentsthereof are incorporated herein by reference as if fully set forthherein.

Without wishing to be bound, it is believed that the effectiveness ofcompounds of Formula I may be attributable to the neighboring groupeffect. More specifically, the supernucleophilicity of the N-hydroxygroup (N-O) may be enhanced by the neighboring group effect of theheteroatoms on the adjacent fused ring. For example, in HOAt,1-hydroxy-7-azabenzotriazole, the supernucleophilicity of the N-hydroxygroup may be enhanced by the neighboring group effect of the pyridinenitrogen atom.

The compounds of Formula I can be prepared by art recognized techniques.The following example is exemplary:

Compounds of the formulae ##STR29## can be prepared by reactinghydrazine with ##STR30## respectively wherein R' is halogen, NH-NH₂ orOR",

R^(") is lower alkyl, such as methyl. This reaction is performed atslightly elevated temperatures, such as 70°-100° C., although thereaction may be performed at temperatures ranging from room temperatureto the boiling point of the solvent.

The reaction is usually run in an organic solvent in which the reactantsare insoluble at room temperature, but in which the reactants andproduct are soluble at slightly elevated temperatures. Examples of thesolvent include ethanol, DMF and the like. In many cases, there is acolor change in the reaction mixture, indicating the formation of theproduct. Work-up, such as removal of the solvent, followed byacidification provides the desired product.

The hydrazino derivative (R¹ =NH-NH₂) of 1 and 2 can be prepared byreacting the corresponding halide, such as chloride or bromide, withhydrazine under substitution reaction conditions. The ether derivative(R¹ =OR") of 1 and 2 can be prepared by reacting the correspondingalcohol with an alkylating reagent, such as Me₂ SO₄ /Na₂ CO₃, underether forming conditions.

Compounds of Formula I, wherein R₃ is hydrogen are also useful forpreparing compounds wherein R₃ is other than hydrogen. These latercompounds can also be prepared by art-recognized techniques. Forexample, compounds of Formula I wherein R₃ is OH are reacted with R₃ Lunder substitution conditions, as indicated hereinbelow ##STR31##

In the above scheme, Q, R₁, R₂, Y, X, R₈, R₉, n and R₃ are as definedhereinabove and L is a leaving group, such as halo (e.g. chloro orbromo). It is preferable that the reaction is run in an inert polarorganic solvent and that the reactants are soluble therein at roomtemperature. It is also preferred that the product is insoluble in thesolvent at room temperature. Examples of the solvent include chloroform,carbon tetrachloride, ethyl ether, dioxane, tetrahydrofuran andmethylene dichloride, and the like. The reaction can take place ateffective temperatures, which may range from the melting point of thesolvent to reflux temperature but it is preferred that the reaction takeplace at about room temperature or at slightly elevated temperatures upto the reflux temperature of the solvent. It is especially preferredthat the reaction take place at room temperature or at slightly elevatedtemperatures, such as up to 60° C.

The N-oxides can be prepared from the compounds of Formula I having anitrogen ring heteroatom in the heteroaryl group. These N-oxides areprepared by art-recognized techniques by oxidation thereof, such as withperacid, e.g., peracetic acid or m-chloroperbenzoic acid.

The following examples further illustrate the invention:

EXAMPLE 1

1-Hydroxy-7-aza-benzotriazole.

To 13.58 g of 2-nitro-3-methoxypyridine was added 26.4 mL of 95%hydrazine, 10.4 mL of water and 15.3 mL of DMF. The mixture wascarefully warmed to about 70° on a hot plate. Spontaneous warming thenset in, the temperature rising to about 80° as the solid dissolved. Thesolution was set aside for 24 hr and then evaporated from a water bathwith the aid of a water aspirator to remove excess hydrazine and water.The dark green residue was cooled in an ice bath, diluted with 50 mL ofwater and acidified to Congo Red with concentrated hydrochloric acid(ca. 17 ml). A yellow-straw-colored solid separated and was filtered andwashed with a little cold water to give 6.25 g (52.1%) of HOAt uponrecrystallization from 75 mL of water gave 5.46 g (45.5%) of nearlycolorless crystals, mp 216°-217° ¹ H NMR (CDCl₃ -DMSO-d₆): δ7.35 (dd, 1,β-H), 8.3 (dd, 1, γ-H), 8.66 (dd, 1, α-H); J.sub.α,.sub.β =4.2 Hz,J.sub.β,γ=8.2 Hz, J.sub.α, γ=1.6 Hz. Evaporation of the filtrate gave anadditional 0.14 g of the pure hydroxy compound so the total yield was5.6 g (46.7%).

EXAMPLE 2

2-(7-Azabenzotriazolyl-1-oxy)-1,1,3,3,-tetramethyluroniumhexafluorophosphate.

To a suspension of 0.88 g of 2-chloro-1,1,3,3,-tetramethyluroniumhexafluorophosphate in 30 mL of methylene dichloride there was added0.43 g of HOAt followed by 0.44 mL of Et₃ N. A clear solution formed andthen a granular solid precipitated. After stirring at room temperaturefor 30 min the solid was filtered, washed twice with 10-mL portions ofmethylene dichloride and dried in air to give 0.79 g (66.4%) of theuronium salt. Recrystallization by solution in 5 mL of acetonitrile atroom temperature, filtration to remove some insoluble crystalline solidand dilution of the filtrate with CH₂ Cl₂ to a total volume of 25 mLgave 0.51 g (44.6%) of the pure uronium salt as shiny white crystals,darkens at 180° melts with decomposition at 190°-194° (gas); ¹ H NMR(DMSO-d₆); δ3.2 (d, 12, CH₃ N), 8.0 (dd, 1, β-H), 8.45 (dd, 1, γ-H), 8.9(dd, 1, α-H); J.sub.α,.sub.β =4.4 Hz, J.sub.β,γ =8.4 Hz, J.sub.α,.sub.δ=l.8 Hz.

Anal:

Calcl for C₁₀ H₁₅ F₆ N₆ OP: C, 31.58, A, 3.97, N, 22.09

Found: C, 31.47, H, 3.94, N, 22.12.

EXAMPLE 3

1-Hydroxy-4-aza-benzotriazole.

To a suspension of 14.3g of 2-chloro-3-nitropyridine in 90 mL ofanhydrous ethanol was slowly added over 2-3 min with swirling 18 mL of95% hydrazine. A new solid took the place of the chloro compound asspontaneous warming occurs. After 20 min at room temperature, filtrationand washing with ethanol gave 15 g of 2-hydrazino-3-nitropyridine as astraw-yellow solid. To 4 g of the crude hydrazine suspended in 24 mL ofanhydrous ethanol was added 16 mL of 95% hydrazine, and the mixture waswarmed on a hot plate until the solid dissolved to give a deepred-colored solution and a spontaneous reaction set in with bubbling.The mixture was removed from the source of heat and the reaction allowedto proceed. After 5 min, the mixture was again heated to the boilingpoint and removed from the hot plate for 5 min. The mixture was treatedtwice more in this manner after which the deeply-colored solution hadbecome lighter in color (reddish yellow). The solution was evaporated ina warm bath with a water aspirator to a brown oily material which wasdissolved in 12 mL of water and acidified (Congo Red) with conc. HCl.Recrystallization from water (Norite) gave 1.10 (31%) of thehydroxytriazole as yellow-colored crystals, mp 203°-211° dec.

EXAMPLE 4

7-Azabenzotriazolyl-1-oxy-trispyrrolidinephosphonium hexafluorophospate.

To a solution of tris-pyrrolidinophosphine (˜0.05 moles) dissolved intetrahydrofuran is added HOAt (˜0.05 moles) triethylamine (˜0.05 moles)and carbon tetrachloride (˜0.1 moles). After stirring at about -30° C.,potassium hexafluorophosphate (˜0.1 moles) dissolved in water is addedto form the above-identified product. Alternatively, an equimolarmixture of trispyrrolidonephosphine oxide and phosphorus oxychloridecould be substituted for the trispyrrolidinophosphine and carbontetrachloride in the above procedure to afford the above-identifiedcompound.

EXAMPLE 5

1-Hydroxy-4-methoxy-7-azabenzotriazole.

The above-identified compound is prepared from3,4-dimethoxy-2-nitropyridine and hydrazine in accordance with theprocedure described in Example 1.

Alternatively, 4-methoxy-3-halo-2-nitropyridine, wherein halo is chloro,bromo or fluoro, may be reacted with hydrazine as described above toafford the above-identified compound.

EXAMPLE 6

4-N,N-dimethylamino-1-hydroxy-7-azabenzotriazole.

The above-identified compound is prepared by reacting4-N-N-dimethylamino-3-methoxy-2-nitro pyridine with hydrazine inaccordance with the procedure described in Example 1.

Alternatively, 4-N,N-dimethylamino-3-halo-2-nitropyridine is reactedwith hydrazine in accordance with the procedure described in Example 1to afford the above-identified compound.

EXAMPLE 7

1-Hydroxy-6-azabenzotriazole.

3-nitro-4-methoxy pyridine is reacted with hydrazine in accordance withthe procedure of Example 1 to yield the above-identified compound.

Alternatively, 3-nitro-4-halo pyridine wherein halo is chloro, fluoro orbromo may be reacted with hydrazine to form the above-identifiedcompound.

EXAMPLE 8

1-Hydroxy-5-azabenzotriazole.

By reacting either 3-methoxy-4-nitro pyridine or 3-halo-4-nitro pyridine(wherein halo is chloro, bromo or fluoro) with hydrazine in accordancewith the procedure of Example 1, the title compound is prepared.

EXAMPLE 9

1-hydroxy-7-aza-1H-indazole

A solution of Na₂ CO₃ 0.10 H₂ O (7.3 mmol) in H₂ O (10 ml) is emulsifiedunder vigorous stirring at about room temperature or slightly elevatedtemperature with a solution of 2-nitro-3-methoxypyridine (4.25 mmol) andtetrabutylammonium bromide (10.06 mmol) as phase transfer catalyst inmethylene chloride (˜20mL). 2-phenyl-5(4H)-oxazolone (˜60 mmol) is addedin several portions during one hour. The layers are separated and theaqueous phase is washed with CH₂ Cl₂. The combined organic solutions aredried with Na₂ SO₄ and is evaporated under reduced pressure. The residueis chromatographed in silica gel starting with petroleum ether to whichmethylene chloride is gradually added. After recrystallizing, thecomplex is placed in refluxing methanol to which a catalytic amount ofp-toluene sulfonic acid has been added. The sample is refluxedovernight. After cooling and evaporation of the solvent, theabove-identified product is isolated.

EXAMPLE 10

1-Hydroxy-7-azabenzo-1H-imidazole.

To a warm solution 2-nitro-3-formamido pyridine in ether is added alc.(NH₄)₂ S to afford the above-identified compound.

EXAMPLE 11

1-Hydroxy-1-H-pyrrolo [2,3-b]pyridine.

The above product is prepared using the methodology described inSynthesis 1983, 537-38.

A mixture of poly-4-vinylpyridine in tetrahydrofuran is dried byazeotropic distillation. 1-H-pyrrolo [2,3-b]pyridine is introduced, thena solution of dibenzoyl peroxide in tetrahydrofuran is added dropwise.After 24 hours at room temperature, the mixture is heated under refluxfor a few hours. The cooled mixture is filtered and the polymer iswashed with dichloromethane. The combined organic phases are evaporatedunder reduced pressure to leave a residue which is diluted with CH₂ Cl₂and washed with 10% Na₂ CO₃ solution. The organic phase is dried withmagnesium sulfate and concentrated and recrystallized to give a solidwhich is dissolved in ether. To the ether solution containing the solidis added potassium methoxide in methanol. The mixture is allowed to bestirred at room temperature for at least 24 hours, and then isconcentrated under reduced pressure and the residual white phase ispartitioned between water and ether. The organic phase is separated, 5%HCl is added, and the precipitate formed is isolated and washed withether. The solid is dissolved in 5% sodium carbonate solution andextracted with fresh ether. Drying and concentration of the ether layersgives the above-identified compound.

EXAMPLE 12 ##STR32##

Using the procedure described in Example 11 and using ##STR33## as thestarting amine, the above-identified compound can be prepared.

EXAMPLE 13 ##STR34##

Using the procedure described in Example 11 and using ##STR35## as theamine reactant the above-identified compound is prepared.

EXAMPLE 14

1-hydroxy-2-oxo-2,3-dihydro-7-azaindole.

A warm solution of 2-nitro-3-methoxyprydine in tetrahydrofuran wasreacted with t-butyl ethyl malonate. The resulting product is nextacidified with concentrated hydrochloric acid and then heated anddecarboxylated to form ethyl 3-([2-nitropyridyl])propanate. Thepropanate is reacted with sodium borohydride over palladium on charcoalor ammonium sulfide to afford the above-identified compound.

EXAMPLE 15 ##STR36##

3-hydroxy-2-nitropyridine is treated with potassium hydroxide and theproduct thereof in turn is reacted with ethyl 2-bromoacetate underWilliamson ether conditions to form ##STR37## which in turn is reactedwith ammonium sulfide or NaBH₄ /PdC to afford the above-identifiedcompound.

EXAMPLE 16 ##STR38##

Using the procedure in Example 15 and substituting3-mercapto-2-nitropyridine for 3-hydroxy-2-nitropyridine, the abovecompound is prepared.

EXAMPLE 17 ##STR39##

Substituting 3-methylamino-2-nitropyridine for3-hydroxy-2-nitropyridine, and using the procedure of Example 15, theabove-identified compound is prepared.

EXAMPLE 18

1-hydroxy-4-t-butyl-7-azabenzotriazole.

The above-identified compound is prepared by reacting2-nitro-3-methoxy-4-t-butylpyridine with hydrazine in accordance withthe procedure described in Example 1.

EXAMPLE 19 ##STR40##

The title compound is prepared by reacting 2-nitro-3-methoxyfuran withhydrazine in accordance with the procedure described in Example 1.

EXAMPLE 20 ##STR41##

Using the procedure described in Example 18 and replacing the pyridinecompound therein with 3-methoxy-2nitrothiophene, the title compound isprepared.

EXAMPLE 21 ##STR42## wherein n is 1 or 2.

The above compound is prepared by coupling ##STR43## with hydrazine inaccordance with the procedure described in Example 1.

EXAMPLE 22 ##STR44## wherein n=0, 1 or 2.

The title compound is prepared by reacting hydrazine with ##STR45## inaccordance with the procedure described in Example 1.

EXAMPLE 23 ##STR46##

The above-identified compound is prepared by reacting2-chloro-1,3-dimethylimidazolinium perfluoroborate with HOAt inaccordance with the procedure described in Example 2.

EXAMPLE 24

1-phenylsulfonyloxy-7-azabenzotriazole

HOAt is reacted with phenylsulfonyl chloride in accordance with theprocedure described in Example 2 to afford the title compound.

EXAMPLE 25

bis-(7-azabenzotraizolyl)carbonate

The title compound is prepared in accordance with the proceduredescribed in Example 2, except that two equivalents of HOAt are reactedwith phosgene.

EXAMPLE 26

bis(7-azabenzotriazolyl)oxalate

The title compound is prepared by following the procedure of Example 2,except that oxalyl chloride is used in place of the hexafluorophosphateand two equivalents of HOAt are used.

EXAMPLE 27

N-oxides or S-oxides of Compounds of Examples 1-26.

The N-oxides of the compounds of Examples 1-22 are prepared by simpleperacid oxidation (e.g., m-chloroperbenzoic acid) of each of saidcompounds.

The N-oxides of the compounds of Examples 23-26 is prepared by firstreacting HOAt with m-chloroperbenzoic acid and then following theprocedure in Examples 23-26, using the N-oxide of HOAt instead of HOAt.

The S-oxides are prepared by reacting the thiophene derivatives withperacid oxidation such as m-chloroperbenzoic acid.

EXAMPLE 28 ##STR47##

A. Approximately one equivalent of the compound ##STR48## is dissolvedin DMF and is reacted slowly with sodium hydride (˜1 eq) in DMF. Theresulting product, compound 11 ##STR49## is formed and is isolated fromthe reaction mixture. Compound 11 is then dissolved in methylenechloride and an excess of trifluoro-acetic acid is added with slightwarming to afford the above-identified compound.

B. Alternatively, to a warm solution of compound in ether is ##STR50##added alcoholic ammonium sulfide to afford the title compound.

EXAMPLE 29

C₆ H₅ CH₂ OCONHC(CH₃)₂ COOAt (Z-Aib-OAt). The N-Benzyloxycarbonylderivative of α-aminoisobutyric acid (Aib; 0.71 g, 3 mmol) was dissolvedin 10 ml of dry THF and the solution cooled in an ice bath and treatedwith 0.411 g (3 mmol) of HOAt followed by 0.618 g of DCC. After 1 h inthe ice bath and 2 h at room temperature 20 ml of EtOAc was added andDCU filtered out. The organic filtrate was washed with 5% citric acid,1M NaHCO₃ and saturated NaCl (2×15-mL each), dried (MgSO₄), and thesolvent removed by Rotovap to give an oil which solidified on theaddition of ether. Recrystallization from CH₂ Cl₂ /hexane gave 0.78 g(73%) of the active ester as a white solid, mp 108°-109° C.; IR (KBr)3328 (NH), 1817 (O-acyl), 1723 (N-acyl), 1700 cm⁻¹ (urethane); ¹ H NMR(CDCl₃) δ1.8 (s, 6H, CH₃), 5.22 (s, 2H, CH₂), 5.6 (s, 1H, NH), 7.1-7.4(m, 6H, aryl), 8.3-8.5 (dd, 1H, α-H), 8.6-8.8 (dd, 1H, γ-H).

Anal. Calcd for C₁₇ H₁₇ N₅ O₄ : C, 57.46; H, 4.79; N, 19.72. Found: C,57.84; H, 4.94; N, 19.46.

Z-Aib-OAt was reacted with p-chloroaniline under amide formingconditions to give the amide, Z-Aib-NHC₆ H₄ Cl-p.

EXAMPLE 30 Z-Phe-OAt

Starting from Z-phenylalamine the preparation followed that which wasgiven for Z-Aib-OAt, the ester being obtained in 76.5% yield as a whitesolid, mp 130°-132° C.; IR (KBr) 3328 (NH), 1823 (O-acyl), 1721(N-acyl), 1700 cm⁻¹ (urethane); ¹ H NMR (CDCl₃) δ3.5 (d, 2H, CH₂),5.1-5.2 (m, 3H, CH, CH₂), 7.2-7.4 (m, 11H, aryl and β-H), 8.4 (d, 1H,α-H), 8.9 (d, 1H, γH).

Anal. Calcd for C₂₂ H₁₉ N₅ O₄ : C, 63.31; H, 4.56. Found: C, 63.59; H,4.78.

EXAMPLE 31 Z-Phg-OAt

Starting from Z-a-phenylglycine the preparation followed that which wasgiven for Z-Aib-OAt, the active ester being obtained in 84.2% yield as afoamy solid, mp 48°-51° C.; IR (KBr) 3318 (NH), 1823 (O-acyl), 1718(N-acyl), 1700 cm⁻¹ (urethane); ¹ H NMR (CDCl₃) δ5.18-5.3 (m, 3H, CH,CH₂), 5.9 (d, 1H, NH), 7.1-7.6 (m, 11H, aryl and β-H), 8.4-8.5 (dd, 1H,α-H), 8.6-8.8 (dd, 1H, γH).

EXAMPLE 32

Comparison of Reactivity of O-Benzoyl Derivatives of HOBt, 7-HOAt and4-HOAt.

The three O-benzoyl derivatives were obtained by reaction of 1 eq ofHOXt wherein X is A or B, with 1 eq of benzoyl chloride and 1 eq of NEt₃(e.g., 68 mg 4-HOAt, 70 mg C₆ H₅ COCl, 50.5 mg NEt₃ in 5 mL of CH₂-Cl₂). After a quick water wash, evaporation gave a solid which wasrecrystallized from toluene/hexane to give the active esters as whitecrystals. To 12.9 mg of t-octylamine in 0.8 g of CDCl₃ was added inthree separate vessels 12 mg of the three O-benzoyl esters. In the caseof O-benzoyl-7-HOAt and O-benzoyl-4-HOAt, acylation was complete withintwo minutes whereas for O-benzoyl-HOBt reaction required about 1 hour.Further experiments using a less reactive nucleophile (such asp-chloroaniline), in accordance with the coupling procedure described inExample 33 showed that 7-aza esters of this type were about twice asreactive as the 4-aza analogs. Both of these esters exceeded thereactivity of the HOBt ester by factors of 10-20.

EXAMPLE 33

General Procedure for Test Couplings. To a solution of 0.37 mmols of aprotected amino acid, 0.33 mmols of HOBt or compounds of Formula I e.g.,HOAt, or other additive, 0.33 mmols of an amino acid ester (or 0.33mmols of the corresponding hydrochloride plus an equivalent amount of atertiary amine) in 1 mL of DMF cooled in an ice bath was added 0.37mmols of DCC or EDC or other condensing agents. For reactions involvingHOAt the disappearance of the yellow color signaled the end of thecoupling process and work up followed soon thereafter. For otheradditives work-up times were arbitrary. Work up consisted of dilutionwith 10 mL of CH₂ Cl₂ followed by 200 mL of water, collection of theorganic layer and extraction with four or five additional 8- to 10-mLportions of CH₂ Cl₂. The combined extracts were washed with two 10-mLportions of 10% HCl, one 10-mL portion of water and two 10-mL portionsof 0.5M NaHCO₃. Drying (MgSO₄) and evaporation of solvent gave theprotected peptide ester which was examined by ¹ NMR for the presence ofdiastereomeric contamination. The results of some couplings are depictedin Table 1.

EXAMPLE 34

Z-Phe-OAt is reacted with valine methyl ester in DMF in accordance withthe procedure described in Example 33 to form Z-Phe-Val.

                                      TABLE 1                                     __________________________________________________________________________    Comparison of HOAt and HOBt in the Coupling.sup.a,b of                        PG--AA.sup.1 --OH + H--AA.sup.2 --OMe + HCl                                   Run                                                                              PG AA.sup.1                                                                           AA.sup.2                                                                         Conditions.sup.c Yield (%)                                                                           DL- (%)                                  __________________________________________________________________________    1  Z  Phg  Val                                                                              HOAt, PS (1 eq), EDC, 9 h                                                                      90.6  <1                                       2  Z  Phg  Val                                                                              HOBt, PS (1 eq), EDC, 9 h                                                                      94.0  3.7                                      3  Z  D-Phg                                                                              Val                                                                              HOAt, PS (1 eq), EDC, 6 h                                                                      70.0  <1                                       4  Z  Phg  Val                                                                              HATU, DIEA (2 eqs), 7 h                                                                        62.8  <1                                       5  Z  Phg  Val                                                                              HBTU, DIEA (2 eqs), 7 h                                                                        56.5  3.8                                      6  Z  Phe--Val                                                                           Ala                                                                              HOAt, NMM (1 eq), EDC, 11/4 h                                                                  72.0  <1                                       7  Z  Phe--Val                                                                           Ala                                                                              HOBt, NMM (1 eq), EDC, 21/4 h                                                                  75.0  4.1                                      8  Z  Phe--Val                                                                           Ala                                                                              HATU, DIEA (2 eqs), 31/2 h                                                                     88.0  <1                                       9  Z  Phe--Val                                                                           Ala                                                                              HBTU, DIEA (2 eqs), 4 h                                                                        72.5  3.6                                      10 Bz Val  Val                                                                              HOAt, NMM (1 eq), EDC, 20 h                                                                    72.0  28.1                                     11 Bz Val  Val                                                                              HOBt, NMM (1 eq), EDC, 20 h                                                                    71.9  45.4                                     12 Bz Val  Val                                                                              DCC, 24 h        70.0  61.5                                     13 Bz Val  Val                                                                              HOAt, DCC, 24 h  87.9  14.4                                     14 Bz Val  Val                                                                              HOBt, DCC, 24 h  85.0  41.9                                     15 Bz Val  Val                                                                              HOAt, DCC, DCM solvent, 24 h                                                                   96.7  <1                                       15 Bz Val  Val                                                                              HATU, NMM (2 eqs), 3 h                                                                         89.8  28.3                                     17 Bz Val  Val                                                                              HBTU, NMM (2 eqs), 31/2 h                                                                      88.0  46.8                                     18 BOC                                                                              Aib  Aib                                                                              HOAt, NMM (1 eq), EDC, 24 h                                                                    99.3  --                                       19 BOC                                                                              Aib  Aib                                                                              HOBt, NMM (1 eq), EDC, 24 h                                                                    65.0  --                                       __________________________________________________________________________     .sup.a Test couplings were carried out by preparing a solution of 0.37        mmol of a protected amino or dipeptide acid, 0.33 mmol of HOAt or HOBt,       0.33 mmol of an amino acid ester (or its hydrochloride plus an equivalent     amount of a tertiary amine) in 1 mL of DMF. The mixture was cooled in an      ice bath and treated with 0.37 mmol of DCC or EDC. For reactions involvin     uronlum salts 0.74 mmol of a tertiary amine was substituted for the HOAt      or HOBt. For HOAt reactions in the absence of excess amino component or       tertiary base disappearance of the yellow color signaled completion of th     coupling process and workup proceeded soon thereafter. In other cases         workup times were arbitrary. Generally stirring was continued in the ice      bath for 11/2-2 h and then at room temperature for the times indicated.       Dilution with 15 mL of CH.sub.2 Cl.sub.2 and 200 mL of water was followed     by extraction with 4-5 10mL portions of CH.sub.2 Cl.sub.2 and washing in      order with 10mL portions of 10% HCl (twice), H.sub.2 O (once), and 0.5 M      NaHCO.sub.3 (twice). Drying and removal of solvent gave the crude peptide     which was examined by .sup.1 H NMR for the presence of diastereomeric         contamination (OMe and/or Me.sub.2 CH--).                                     .sup.b Abbreviations: PG = protecting group, AA.sub.1, AA.sub.2 = amino       acid or dipeptide fragment, PS = proton sponge                                (1,8bis(N,N-dimethylamino)naphthalene), EDC =                                 Nethyl-N(3-dimethylaminopropyl)-carbodilmide hydrochloride, DIEA =            diisopropylethylamine, NMM = Nmethylmorpholine, DCC =                         dicyclohexylcarbodiimide, DCM = dichloromethane, Z = benzyloxycarbonyl, B     = benzoyl, BOC = tbutyloxycarbonyl.                                           .sup.c All reactions were carried out in DMF except where indicated.     

Table 1 illustrates the effectiveness of the compounds of the presentinvention in promoting peptide coupling. This is demonstrated using arepresentative example, such as HOAt. Further, the table illustrates theeffectiveness of compounds of the present invention, such as HOAt, inreducing racemization.

One model system that was used involved coupling of an urethaneprotective derivative of the sensitive non-proteinogenic amino acidα-phenylglycine and comparing the amount of racemization when HOBT andan exemplary compound of the present invention, e.g., HOAt, were used asthe additive. For example, upon treatment of the benzyloxycarbonylderivative with valine methyl ester hydrochloride in the presence ofHOBt and an equivalent of proton sponge or with HBTU and two equivalentsof DIEA, 3.7-3.8% of the DL-diastereomer was formed (runs 2 and 5). Thiswas reduced to less than 1% by substitution of HOAt for HOBt in thesereactions (runs 1 and 4).

A second example, even more promising in view of its relevance tosegment coupling, involved reaction of Z-Phe-Val-OH with alanine methylester. With this system HOBt- or HBTU- coupling (runs 7 and 9) in thepresence of NMM or DIEA gave 3.6-4.1% of the LDL-isomer. Again in thiscase, the use of compounds of the present invention, e.g., HOAt or HATUlowers the extent of racemization to less than 1% (runs 6 and 8).Finally the highly sensitive coupling of benzoylvaline with valinemethyl ester shows that HOAt reduces racemization to about one fourth orone half the level found for comparable HOBt reactions (runs 10-17). Forthis system, even with HOAt, only in a non-polar solvent such asdichloromethane was it possible to effect coupling without detectableracemization.

An example of the accelerated reactivity in the presence of compounds ofthe present invention is illustrated in the following example: ##STR51##

This example involves the coupling of the hindered amino acid α-aminoisobutyric acid (Aib). In the presence of HOBt, this reaction isincomplete after 24 hours (25-35% HOBt ester remaining unreacted)whereas complete coupling is observed with HOAt.

The compounds of the present invention are also useful in promoting thecoupling of an amine with a carboxylic acid or acylating derivativethereof. For example, the reaction of a hindered secondary amine 23 withBOC-Pro-OH is over within 1/2 hr in the presence of HOAt, whereas it isstill incomplete after 24 hours ##STR52## with HOBt. Similarlybenzoylation of the even more hindered trans-amine 24 via EDC along witheither HOBt or HOAt requires about 48 hours in the case of HOBt and only5 hours for HOAt.

Ester-, as well as amide-bond formation can be achieved with thesereagents. In the present study it was found that formation of the methylester of the dipeptide FMOC-Phg-Phe-OH in the presence of HOBt and acatalytic amount of proton sponge occurred with contamination by 20.5%of the D, L-form whereas with HOAt under the same conditions,racemization was not eliminated although it could be reduced to 7.5%. Onthe other hand it was found that with HOAt no base was required and inthat case no significant racemization occurred. A parallel reaction withHOBt alone gave only a trace of the ester under the same conditions.

As indicated above, compounds of the present invention, e.g. HOAt, arealso unique in exhibiting the effect of a built-in indicator which,under appropriate conditions, signals completion of the couplingreaction. For example, the anion of HOAt ##STR53## is yellow. In thepresence of an amino acid or peptide ester, HOAt is converted to itshighly colored yellow anion. As coupling proceeds, the color fades andthen disappears completely. For couplings between unhindered amino acidresidues, this happens within 15-20 min in DMF solution. On the otherhand, current recipes for the use of HOBt as an additive often involvereaction periods of 10-24 hours.

It should be noted that the maximum neighboring group effect (reactivityacceleration and racemization reduction) is observed when the heteroatomin the adjacent fused ring is in the neighboring group position relativeto the (N-O) bond. For example, the neighboring group effect is greaterin HOAt, i.e., ##STR54## than in 1-hydroxy-4-azabenzotriazole, i.e.,##STR55## However, in cases like the latter, reactivity is stillaccelerated, but no racemization reduction is observed. But, there aretimes when only acceleration is needed, such as in step-wise solid phasesynthesis, wherein there is no fear of racemization. Compounds whereinthe heteroatom is not on the neighboring group relative to the N-O bondsatisfy those needs.

The above preferred embodiments and examples are given to illustrate thescope and spirit of the present invention. These embodiments andexamples will make apparent to those skilled in the art otherembodiments and examples. These other embodiments are also exampleswithin the contemplation of the present invention. Therefore, thepresent invention should be limited only by the appended claims.

What is claimed:
 1. A compound of the formula: ##STR56## or N-oxidesthereof or salts thereof wherein D is CR₁₆ or N;E is CR₁₇ or N; A isCR₁₅ or N; G is CR₁₈ or N;provided that one and only one of A, E. D or Gis N; R₁₅, R₁₆, R₁₇ and R₁₈ are independently hydrogen, lower alkyl oran electron donating group; X is CR₇ or N; Y is N or CR₅ ; R₇ and R₅ areindependently hydrogen or lower alkyl; R₃ is SO₂ R₁₄ lower alkylcarbonyl, aryl carbonyl, aryl lower alkyl carbonyl, a positively chargedelectron withdrawing group or AA₁ -BLK₁ ; R₁₄ is lower alkyl, aryl oraryl lower alkyl; BLK₁ is an amino acid protecting group; and AA₁ is anamino acid or peptide less a hydrogen on the N-terminus and an OH on theC-terminus.
 2. The compound according to claim 1 wherein A or G is N. 3.The compound according to claim 1 wherein Y is CH or N and X is CH or N.4. The compound according to claim 1 wherein the electron donating groupis amino, lower alkylamino, diloweralkylamino, hydroxy, lower alkoxy,lower aralkoxy, halo, aryl, mercapto, or lower alkylthio.
 5. Thecompound according to claim 4 wherein the electron donating group islower dialkylamino or lower alkoxy.
 6. The compond according to claim 1having the formula ##STR57## in which Y is N or CH and X is N or CH. 7.The compound according to claim 6 wherein R₃ is OSO₂ R₁₄, loweralkylcarbonyl, aryl carbonyl, or aryl lower alkyl carbonyl.
 8. Thecompound according to claim 6 wherein R₃ is AA₁ -BLK₁.
 9. The compoundaccording to claim 6 wherein R₃ is a positively charged electronwithdrawing group.
 10. The compound according to claim 9 wherein R₃ is##STR58## wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen,lower alkyl or lower alkoxy lower alkyl orR₁₀ and R₁₁ taken togetherwith the nitrogen atom to which they are attached form a heterocyclicring containing up to 6 ring atoms and up to a total of 5 ring carbonatoms or R₁₀ and R₁₂ taken together with the atoms to which they areattached form a heterocyclic ring containing up to 6 ring atoms and upto a total of 5 ring carbon atoms or R₁₂ and R₁₃ taken together with thenitrogen atom to which they are attached form a 5- or 6- memberedheterocyclic ring containing up to a total of 5 ring carbon atoms. 11.The compound according to claim 10 wherein R₁₀, R₁₁, R₁₂ and R₁₃ areindependently lower alkyl, hydrogen or lower alkoxy lower alkyl.
 12. Thecompound according to claim 11 wherein R₁₀, R₁₁, R₁₂ and R₁₃ areindependently lower alkyl.
 13. The compound according to claim 12wherein R₁₀, R₁₁, R₁₂ and R₁₃ are methyl.
 14. The compound according toclaim 10 wherein R₃ is ##STR59## and m is 0 or
 1. 15. The compoundaccording to claim 14 wherein R₁₁ and R₁₂ are independently hydrogen,methyl, ethyl, propyl, butyl, pentyl or CH₂ --CH₂ O--CH₂ --CH₃.
 16. Thecompound according to claim 6 wherein R₃ is ##STR60## wherein U isN-ALK, CH₂ or O, and ALK is lower alkyl or hydrogen.
 17. The compoundaccording to claim 16 wherein U is CH₂.
 18. The compound according toclaim 6 wherein the compound has the formula: ##STR61##
 19. The compoundaccording to claim 18 wherein R₃ is OSO₂ R₁₄, lower alkylcarbonyl, arylcarbonyl, or aryl lower alkyl carbonyl.
 20. The compound according toclaim 18 wherein R₃ is AA₁ -BLK₁.
 21. The compound according to claim 18wherein R₃ is a positively charged electron withdrawing group.
 22. Thecompound according to claim 21 wherein R₃ is ##STR62## wherein R₁₀, R₁₁,R₁₂ and R₁₃ are independently hydrogen, lower alkyl or lower alkoxylower alkyl orR₁₀ R₁₁ taken together with the nitrogen atom to whichthey are attached from a heterocyclic ring containing up to 6 ring atomsand up to a total of 5 ring carbon atoms or R₁₀ and R₁₂ taken togetherwith the atoms to which they are attached form a heterocyclic ringcontaining up to 6 ring atoms and up to a total of 5 ring carbon atomsor R₁₂ and R₁₃ taken together with the nitrogen atoms to which they areattached form a 5- or 6- membered heterocyclic ring containing up to atotal of 5 ring carbon atoms.
 23. The compound according to claim 22wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently lower alkyl, hydrogen orlower alkoxy lower alkyl.
 24. The compound according to claim 23 whereinR₁₀, R₁₁, R₁₂ and R₁₃ are independently lower alkyl.
 25. The compoundaccording to claim 24 wherein R₁₀, R₁₁, R₁₂ and R₁₃ are methyl.
 26. Thecompound according to claim 22 wherein R₃ is ##STR63## and m is 0 or 1.27. The compound according to claim 26 wherein R₁₁ and R₁₂ areindependently hydrogen, methyl, ethyl, propyl, butyl, pentyl or CH₂--CH₂ O--CH₂ --CH₃.
 28. The compound according to claim 18 wherein R₃ is##STR64## wherein U is N-ALK, CH₂ or O, and ALK is lower alkyl orhydrogen.
 29. The compound according to claim 28 wherein U is CH₂.
 30. Acompound of the formula: ##STR65## or N-oxides thereof or salts thereofwherein D is CR₁₆ or N;E is CR₁₇ or N; A is CR₁₅ or N; G is CR₁₈ orN;and at least two of A, E, D and G are N; R₁₅, R₁₇ and R₁₈ areindependently hydrogen, lower alkyl or an electron donating group; R₃ ishydrogen, lower alkyl carbonyl, aryl carbonyl, aryl lower alkylcarbonyl, SO₂ R₁₄, a positively charged electron withdrawing group orAA₁ -BLK₁ ; R₁₄ is lower alkyl, aryl, or aryl lower alkyl; BLK₁ is anamino acid protecting group, and AA₁ is an amino acid or peptide less ahydrogen on the N-terminus and an OH on the C-terminus.
 31. The compoundof claim 30 wherein two of A, D, E and G are N.
 32. The compound ofclaim 30 wherein the electron donating group is amino, lower alkylamino,diloweralkylamino, hydroxy, lower alkoxy, lower aralkoxy, halo, aryl,mercapto, or lower alkylthio.
 33. The compound of claim 32 wherein theelectron donating group is lower dialkylamino or lower alkoxy.
 34. Thecompound of claim 30 wherein R₃ is hydrogen.
 35. The compound of claim30 wherein R₃ is OSO₂ R₁₄, lower alkylcarbonyl, aryl carbonyl, or aryllower alkyl carbonyl.
 36. The compound of claim 30 wherein R₃ is BLK₁-AA₁.
 37. The compound of claim 30 wherein R₃ is a positively chargedelectron withdrawing group.
 38. The compound of claim 37 wherein R₃ is##STR66## wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen,lower alkyl or lower alkoxy lower alkyl orR₁₀ and R₁₁ taken togetherwith the nitrogen atom to which they are attached form a heterocyclicring containing up to 6 ring atoms and up to a total 5 ring carbon atomsor R₁₀ and R₁₂ taken together with the atoms to which they are attachedform a heterocyclic ring containing up to 6 ring atoms and up to a totalof 5 ring carbon atoms or R₁₂ and R₁₃ taken together with the nitrogenatom to which they are attached form a 5- or 6- membered heterocyclicring containing up to a total of 5 ring carbon atoms.
 39. The compoundof claim 38 wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently lower alkyl,hydrogen or lower alkoxy lower alkyl.
 40. The compound of claim 38wherein R₃ is ##STR67## and m is 0 or
 1. 41. The compund according toclaim 30 wherein R₃ is ##STR68## wherein U is N-ALK, CH₂ or O, and ALKis lower alkyl or hydrogen.
 42. A compound of the formula: ##STR69## orN-oxides thereof or salts thereof; whereinX is CR₇ or N; Y is CR₅ or N;with the proviso that X and Y cannot both represent N simultaneously; R₅and R₇ are independently hydrogen or lower alkyl; D is CR₁₆ or N; E isCR₁₇ or N; A is CR₁₅ or N; G is CR₁₈ or N;with the further proviso thatone and only one of A, E, D and G is N; R₁₅, R₁₆, R₁₇ and R₁₈ areindependently hydrogen, lower alkyl or an electron donating group; R₃ ishydrogen, SO₂ R₁₄ lower alkyl carbonyl, aryl carbonyl, aryl lower alkylcarbonyl, a positively charged electron withdrawing group or AA₁ -BLK₁;R₁₄ is lower alkyl, aryl or aryl lower alkyl; BLK₁ is an amino acidprotecting group andAA₁ is an amino acid or peptide less an hydrogen onthe N-terminus and an OH on the C-terminus.
 43. The compound accordingto claim 42 wherein R₃ is hydrogen.
 44. The compound according to claim42 having the formula: ##STR70##
 45. The compound according to claim 44wherein Y is CR₅ and X is N.
 46. The compound according to claim 45wherein Y is CH.
 47. The compound according to claim 44 wherein X is CR₇and Y is N.
 48. The compound according to claim 47 wherein X is CH. 49.The compound according to claim 44 wherein X is CR₇ and Y is CR₅. 50.The compound according to claim 49 wherein X and Y are both CH.
 51. Acompound of the formula: ##STR71## wherein D is CR₁₆ or N;E is CR₁₇ orN; A is CR₁₅ ; G is CR₁₈ ;provided that one and only one of D and E isN; R₁₅, R₁₆, R₁₇ and R₁₈ are independently hydrogen, lower alkyl or anelectron donating group, R₃ is hydrogen, SO₂ R₁₄ lower alkyl carbonyl,aryl carbonyl, aryl lower alkyl carbonyl, a positively charged electronwithdrawing group or AA₁ -BLK₁ ; R₁₄ is lower alkyl, aryl or aryl loweralkyl; BLK₁ is an amino acid or peptide less an hydrogen on theN-terminus and an OH on the C-terminus.
 52. The compound according toclaim 51 wherein D is N.
 53. The compound according to claim 51 whereinE is N.
 54. The compound according to claim 51 wherein D is N and A andG are CH.
 55. The compound according to claim 54 wherein R₃ is hydrogen.56. The compound according to claim 51 wherein E is N and A and G areCH.
 57. The compound according to claim 56 wherein R₃ is hydrogen.
 58. Acompound of the formula: ##STR72## or N-oxides thereof or salts thereofwherein D is CR₁₆ or N;E is CR₁₇ or N; A is CR₁₅ or N; G is CR₁₈ orN;provided that one and only one of D, E, A, and G is N; at least one ofR₁₅, R₁₆, R₁₇ and R₁₈ is an electron donating group, and the remainderis independently hydrogen, lower alkyl or an electron donating group; Xis CR₇ or N; Y is N or CR₅ ; R₇ and R₅ are independently hydrogen orlower alkyl; R₃ is hydrogen, SO₂ R₁₄ lower alkyl carbonyl, arylcarbonyl, aryl lower alkyl carbonyl, a positively charged electronwithdrawing group or AA₁ -BLK₁ ; R₁₄ is lower alkyl, aryl or aryl loweralkyl; BLK₁ is an amino acid protecting group and AA₁ is an amino acidor peptide less a hydrogen on the N-terminus and an OH on theC-terminus.
 59. The compund according to claim 58 wherein R₃ ishydrogen.
 60. The compound according to claim 58 wherein R₃ is AA₁-BLK₁.
 61. The compound according to claim 58 wherein R₃ is a positivelycharged electron withdrawing group.
 62. The compound according to claim58 wherein A or G is N.
 63. The compound according to claim 58 wherein Yis CH or N and X is CH or N.
 64. The compound according to claim 58wherein the electron donating group is diloweralkylamino, lower alkoxy,lower aralkoxy, halo, aryl or lower alkylthio.
 65. The compoundaccording to claim 64 wherein the electron donating group is lowerdialkylamino or lower alkoxy.
 66. The compound according to claim 58having the formula: ##STR73## wherein R₁₅ is lower alkyl or an electrondonating group, and R₁₇ is lower alkyl, hydrogen or an electron donatinggroup.
 67. The compound according to claim 66 wherein R₃ is hydrogen.68. The compound according to claim 66 wherein R₃ is OSO₂ R₁₄, loweralkyl carbonyl, aryl carbonyl or aryl lower alkyl carbonyl.
 69. Thecompound according to claim 66 wherein R₃ is AA₁ -BLK₁.
 70. The compoundaccording to claim 66 wherein R₃ is a positively charged electronwithdrawing group.
 71. The compound according to claim 70 wherein R₃ is##STR74## wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen,lower alkyl or lower alkoxy lower alkyl orR₁₀ and R₁₁ taken togetherwith the nitrogen atom to which they are attached form a heterocyclicring containing up to 6 ring atoms and up to a total of 5 ring carbonatoms or R₁₀ and R₁₂ taken together with the atoms to which they areattached form a heterocyclic ring containing up to 6 ring atoms and upto a total of 5 ring carbon atoms or R₁₂ and R₁₃ taken together with thenitrogen atom to which they are attached form a 5- or 6- memberedheterocyclic ring containing up to a total of 5 ring carbon atoms. 72.The compound according to claim 71 wherein R₁₀, R₁₁, R₁₂ and R₁₃ areindependently lower alkyl, hydrogen or lower alkoxy lower alkyl.
 73. Thecompound according to claim 72 wherein R₁₀, R₁₁, R₁₂ and R₁₃ areindependently lower alkyl.
 74. The compound according to claim 73wherein R₁₀, R₁₁, R₁₂ and R₁₃ are methyl.
 75. The compound according toclaim 71 wherein R₃ is ##STR75## wherein m is 0 or
 1. 76. The compoundaccording to claim 70 wherein R₃ is ##STR76## wherein U is N-ALK, CH₂ orO, and ALK is lower alkyl or hydrogen.
 77. The compound according toclaim 66 wherein R₁₅ is an electron donating group and R₁₇ is hydrogenor an electron donating group.
 78. The compound according to claim 77wherein the electron donating group is diloweralkylamino or loweralkoxy.
 79. An N-oxide of a compound of the formula: ##STR77## or a saltthereof, whereinD is CR₁₆ or N; E is CR₁₇ or N; A is CR₁₅ or N; G isCR₁₈ or N;provided that one and only one of A, E,D or G is N; R₁₅, R₁₆,R₁₇ and R₁₈ are independently hydrogen, lower alkyl and an electrondonating group, X is CR₇ or N; Y is CR₅ or N; R₇ and R₅ areindependently hydrogen or lower alkyl; R₃ is hydrogen, SO₂ R₁₄ , loweralkyl carbonyl, aryl carbonyl, lower alkyl carbonyl, a positivelycharged electron withdrawing group or BLK₁ -AA₁ ; R₁₄ is lower alkyl,aryl or aryl lower alkyl; BLK₁ is an amino acid protecting group and AA₁is an amino acid or peptide less a hydrogen on the N-terminus and an OHon the C-terminus.
 80. The compound according to claim 79 wherein R₃ ishydrogen.
 81. The compund according to claim 79 wherein R₃ is OSO₂ R₁₄,lower alkyl carbonyl, aryl carbonyl, or lower alkyl carbonyl.
 82. Thecompound according to claim 79 wherein R₃ is a positively chargedelectron withdrawing group.
 83. The compound according to claim 82wherein R₃ is ##STR78## wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independentlyhalogen, lower alkyl or lower alkoxy lower alkyl orR₁₀ and R₁₁ takentogether with the nitrogen atom to which they are attached form aheterocyclic ring containing up to 6 ring atoms and up to a total of 5ring carbon atoms or R₁₀ and R₁₂ taken together with the atoms to whichthey are attached form a heterocyclic ring containing up to 6 ring atomsand up to a total of 5 ring carbon atoms or R₁ and R₁₃ taken togetherwith the nitrogen atom to which they are attached form a 5- or6-membered heterocyclic ring containing up to a total of 5 ring carbonatoms.
 84. The compound according to claim 79 wherein A or G is N. 85.The compound according to claim 79 wherein Y is CH or N and X is CH orN.
 86. The compound according to claim 79 having the formula: ##STR79##87. The compound according to claim 86 wherein R₃ is hydrogen.
 88. Thecompound according to claim 86 wherein R₃ is OSO₂ R₁₄, lower alkylcarbonyl, aryl carbonyl, or lower alkyl carbonyl.
 89. The compoundaccording to claim 86 wherein R₃ is a positively charged electronwithdrawing group.
 90. The compound according to claim 89 wherein R₃ is##STR80## wherein R₁₀, R₁₁, R₁₂ and R₁₃ are independently hydrogen,lower alkyl or lower alkoxy lower alkyl orR₁₀ and R₁₁ taken togetherwith the nitrogen atom to which they are attached form a heterocyclicring containing up to 6 ring atoms and up to a total of 5 ring carbonatoms or R₁₀ and R₁₂ taken together with the atoms to which they areattached form a heterocyclic ring containing up to 6 ring atoms and upto a total of 5 ring carbon atoms or R₁₂ and R₁₃ taken together with thenitrogen atom to which they are attached form a 5- or 6- memberedheterocyclic ring containing up to a total of 5 ring carbon atoms. 91.The compound according to claim 1 of the formula ##STR81## wherein R₃ is##STR82## wherein U is CH₂, O or N-ALK and ALK is H or lower alkyl. 92.The compound according to claim 1 of the formula: ##STR83## wherein U isCH₂, O or N-ALK and ALK is H or lower alkyl.
 93. The compound accordingto claim 91 wherein U is CH₂.